The invention relates to a device for analyzing immunoassays with a liquid assay medium which is limited by at least one boundary surface of a solid body, wherein first reaction agents are dissolved in the assay medium and are labeled with a luminophore or different luminophores and second reaction agents are bonded to the boundary surface within a boundary layer of the assay medium, and wherein for a quantitative detection of the first reaction agents with the aid of light rays emitted by a transmitter, an evanescent field is generated through which luminophore-labeled first reaction agents that are bonded to the second reaction agents are optically excited and emit fluorescent rays and/or phosphorescent rays that can be detected in a receiver.
The field of medical diagnostics, specifically immunological diagnostics, is based to a high degree on the ELISA (enzyme-linked immunosorbent assay). A recent overview of immunoassays can be found in Hage, Anal. Chem., Vol. 71, pages 294R–304R, 1999. Two features characterize an ELISA. A first reaction agent is labeled with an enzyme and is dissolved in an assay medium. A second reaction agent is bonded to a solid phase, wherein the solid phase is formed by a boundary surface of a solid body that limits the assay medium.
Standardized plastic panels, frequently made of polystyrene, which contain 96 wells are primarily used for the solid phase. The plastic well surface bonds proteins, which form the second reaction agents, through adsorption in the nanogram range. This amount is sufficient for an immunological detection. A bonding reaction with the first, enzyme-labeled reaction agent present in the solution leads to the bonding of the enzyme to the solid phase. The bonded enzyme is made visible through adding a chromogen substrate that is specific for this enzyme. Subsequently, the resulting colored product can be evaluated optically.
A number of technical options exist for labeling the first reaction agent, mostly an immunoglobulin, with an enzyme. Peroxidase or alkaline phosphatase is commonly used for the labeling.
Excellent results can be obtained with ELISAs with respect to sensitivity and specificity. The achievable detection limits are in the nanogram range or below.
Assays based on this principle are realized in the most varied forms and are used for detecting antigens or anti-bodies, depending on the problem definition.
However, one significant disadvantage of the ELISA is the handling of the tests because different reagents must be added successively to the wells and must also be removed again. The total of the various pipetting, washing, and incubation steps is different from assay to assay and can number ten or more. For that reason, ELISAs are time-consuming and their performance is very involved operationally. ELISAs must be carried out with great precision by specially trained personnel to achieve good results.
Another disadvantage of the ELISA is the time required for an assay, which is determined by the sum of the incubation and washing steps and normally lasts from one to several hours.
A device for analyzing immunoassays is known from U.S. Pat. No. 3,939,350, for which a transparent disk can be inserted between a prism and a container holding the assay medium.
By means of a laser, transmitted light rays are beamed at the total reflection angle onto the disk, thereby creating an evanescent field in the border region of the assay medium, in the disk area.
The first reaction agent in the solution is labeled with a luminophore. The second reaction agent is bonded to the surface of the disk. If the luminophore-labeled reaction agent in the solution bonds to the surface of the disk, it can subsequently be excited by the evanescent field of a totally reflected light ray and can emit fluorescent radiation. This fluorescent radiation is determined quantitatively and is directly proportional to the bonded luminophore-labeled reaction agent and is thus directly proportional to the amount of the originally existing reaction agent in the solution.
Since only the luminophore bonded to the surface is positioned in the field of evanescence of the laser beam, only this bonded luminophore is optimally excited and emits photons. A luminophore in the solution that is not bonded is not positioned in the field of evanescence of the light beam, is therefore not excited and consequently also does not emit photons. This arrangement therefore permits the quantitative determination of bonded luminophore in the presence of non-bonded luminophore.
However, the disadvantage in this case is the mechanically complicated design of the device. In particular the preparation of the disk and the subsequent insertion of the disk between prism and container are extremely time-consuming. In addition, the preparation can only be carried out by qualified personnel.